To investigate the genetic evolution and antigenic variation of H9N2 subtype avian influenza virus (AIV) in Anhui Province, China, this study collected oropharyngeal swab samples from poultry in live bird markets, large-scale poultry farms, and slaughterhouses in both urban and rural areas throughout 2024. Following virus isolation and identification, four H9N2 AIV strains isolated in 2024 were selected for whole-genome sequencing. These sequences, along with all publicly available H9N2 AIV sequences from Anhui Province, were analyzed for homology, phylogenetic relationships, key amino acid mutations, glycosylation site changes, and antigenic variation across eight gene segments (PB2, PB1, PA, HA, NP, NA, M and NS). A total of 1033 swab samples were collected, from which 334 H9N2 AIV strains were identified, resulting in an isolation rate of 32.33%. Among the four sequenced isolates from 2024, the PB1, PA, and HA genes exhibited substantial sequence divergence (88.65%-98.05%), while the PB2, NP, NA, M and NS genes were more conserved (92.19%~99.08%). Phylogenetic analysis of the HA gene revealed that two chicken-derived and one duck-derived strains belonged to sub-clade B4.7.2 of the B lineage, while one goose-derived strain belonged to sub-clade B4.7.3. The PB2, PB1, PA, NP, NA, M and NS genes of the 2024 isolates were genetically close to recent human H9N2 isolates, with the PA gene of one February 2024 chicken-derived strain closely related to a wild bird-derived H9N2 virus. Analysis of key amino acids revealed notable polymorphisms in the 130-loop, 150-loop, and 190-helix regions of the HA protein compared to reference strains. Additionally, the HA protein of the goose-derived strain showed a potential loss of N-glycosylation at positions 289~291, which warrants further attention. Antigenic evaluation indicated that all 2024 isolates belonged to the currently dominant antigenic group, although the goose-derived strain exhibited significant antigenic drift. In conclusion, the 2024 H9N2 AIV strains circulating in Anhui Province demonstrated high genetic diversity across hosts. Mutations in the receptor-binding domain of the HA protein and evidence of antigenic drift suggest a continued risk of transmission and adaptation to both poultry and humans.